President: ForensiGen, LLC Founder and President The ... · •Match –Peaks between the compared...
Transcript of President: ForensiGen, LLC Founder and President The ... · •Match –Peaks between the compared...
Maher “Max” Noureddine, PhD, MS, D-ABCPresident: ForensiGen, LLC
Founder and President
The Institute for Advanced Career Development
3/19/2015
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Forensic DNA and Serology Evidence: Science, Justice, and the Gaps in Between
Crime Scene: Biological Evidence Processing
Evidence Collection and Preservation
Field Testing Laboratory Testing
Identification
What is it? Who is it from?
Suspect (s)
Cold Hit
CODISDatabase
Serology DNA
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What is it? Forensic Serology
Blood
Saliva
Semen
Others??
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Ex: 450nm Orange filter
Possible Semen Stain
Source: PD
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Crime Scene: Biological Evidence Processing
Forensic Serology
Presumptive Tests:
Confirmatory Tests:
‐Used for preliminary screening
‐Used for confirmation / validation (alone or in conjunction with a presumptive test)
‐Prone to false positive and false negative
‐False positive is rare, but false negative can happen
‐Test is mostly chemical in nature
‐Test is chemical, biological, or physical in nature
‐Look for a color change or luminescence (usually a catalyzed oxidation reaction)
‐Identify/detect UNIQUE characteristics using various technologies
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Case 7
Greg Taylor: Convicted in Jacquetta Thomas’ murder in1993exonerated in 2010
Forensic Serology
Oxidation of PhenolphthalinThe Kastle‐Meyer Test
Phenolphthalin Phenolphthalein
Pink
Iron
Colorless
Source: PD
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Forensic Serology
Hemachromagen Crystal Assay (Takayama Assay)
Confirmatory Blood Test
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Lab’s final conclusions:
“Semen was found on the external genitalia swabs”.
However! The Lab’s report (detailed section) also states:
The acid phosphatase test was Inconclusive. (P)
The P30 (or PSA) was weak positive. (P?)
Microscopic exam for the presence of sperm was negative. (C)
And, DNA analyses were “negative” for male DNA !!!9
Case 8
Forensic Serology
The Prostate gland
Source: VD Graaf
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Forensic Serology
The Skene's glands (aka the lesser vestibular glands, periurethral glands)
Skene's glands
Source: VD Graaf
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Forensic Serology
Acrosomal cap: clear to pink
Acrosome: dark pink to red
Midpiece: green to blue
Tail: green to blue
Sperm IdentificationChristmas Tree Test
Source: PD
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Population O A B ABUS Whites 45.3% 41.3% 9.9% 3.5%
US Blacks 49.1% 26.5% 20.1% 4.3%
Chinese 43.9% 27.0% 23.3% 5.8%
Bolivian 93.1% 5.3% 1.6% 0.1%
Blood Typing and Identification
The Science of Serology and ForensicsSource: NCBI
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Source: Innocence Project
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Genetic Principles and DNA as a
Forensic Tool
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Deoxyribonucleic Acid (DNA)Source: PD
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James Watson
Rosalind Franklin
Francis Crick
1870Johann Friedrich Miescher
1953
DNA Discovery
1962
1944Avery and Griffin
Source: Wikipedia
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DNA in Living ThingsSource: PD
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DNA in the CellSource: PD
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DNA is packed inside a compartment called “The Nucleus”
Human DNA: Chromosomes
Human Chromosomes
23 Pairs of chromosomes in every human cell (with few exceptions): One copy from Mom, one copy from Dad
Source: PD
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Fruit Fly: 8
Broccoli: 18
Cat: 38
Dog: 78
Goldfish: 94
Ferns: 1200+
Human Chromosomes
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Chromosomes in Living Organisms
# of Chr in:
Cell Division Requires DNA Replication
DNA
DNA
DNA
1 cell
2 cells
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23 chromosomes in sperm
23 chromosomes in egg
46 chromosomes
Fert
iliza
tion
DNA in the Cell
Every Chromosome
is a continuous
double strand of
DNA…Think of it as a
long string of the 4
DNA molecules:
(A, G, C, and T) !
GAAACTTGCA…..CTTT GAACGT…..
Source: PD
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A gene
A switch
...
DNA in the Cell
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Source: PD
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Chromosome 15
What is a Locus or Loci?A region or sequence on a chromosome that is responsible for a trait or a genetic characteristic (e.g. a specific location of a gene for hair color).
What is an Allele?An allele is a variant of a gene or genetic sequence at a given genetic locus
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R
Homozygous R
R R B B B
Heterozygous R,B Homozygous B
Allele Inheritance for Locus “Cat Allergy1”
Mike Judy
Anna Paul Tony Phil
a b a c
caabcbaa
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Chromosome 15
Forensic DNA Analysis
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Locus (Loci): Location on a Chromosome
5 8Chromosome 16
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Locus = Marker
Marker D16S10
Remember: one copy from momone copy from dad
…AAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCCATTTGAC GATA GATA GATA GATAGATA CCCTCTTC…
…AAGTTCATGTAAATGTAGTCCCAGTCCAGTCAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCCATTTGAC GATA GATA GATA GATAGATA GATA GATA GATA CCCTCTTC…
Short Tandem Repeats (STRs) in DNA
Chromosome 16 Marker D16S539
5 Repeats of “GATA”
8 Repeats of “GATA”
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GAATT GAATT
GAATT GAATT GAATT GAATT
Short Tandem Repeats
Allele1
Allele2
2 repeats
5 repeats
Example: a locus on Chromosome 10
Number of STRs Provides Uniqueness
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GAATT
35413244# repeats
Chr 8(D8S1179)
Chr 21(D21S11)
Chr 7(D7S820)
Chr 13(D13S317)
Chromosome #Marker Name
Short Tandem Repeats (STRs) in DNA
GenotypeOr “Profile”:
4 , 4 2 , 3 1 , 4 5 , 3
or “4”32
GAATT GAATT
GAATT GAATT GAATT GAATT GAATT
Allele1
Allele2
2 repeats
5 repeats
Restriction Fragment Length Polymorphism
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Gel Electrophoresis
DNA fragment migration through a gel
Source: PD
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-Each white band is a DNA fragment-Each column or “lane” is a DNA sample from an individual
Dire
ctio
n of
DN
A m
ovem
ent
Who is the possible Biological Child?
(14)
(12)
(11)
(9)
(8)
(7)
(6)
(5)
(4)
(3)
(13)
(10)
TH01alleles
Alleleladder
Mother Father C1 C2 C3
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(12)
(13)(13)
(10)(10)(10)
(6)
(5)
(6)
(3)
Restriction Fragment Length Polymorphism
-Requires lots of cells (5,000-30,000 cells)
-Requires intact DNA
-Requires a way to distinguish size differences
-Requires the use of radioactive material
Sir Alec Jeffreys, a professor of genetics at the
University of Leicester, developed DNA fingerprinting.
Used as a criminal tool for the first time in 1988 to
convict Colin Pitchfork in the rape/murder of Lynda Mann
and Dawn Ashworth in Narborough, England
Source: Wikipedia
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Modern Forensic DNA Analysis
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Polymerase Chain Reaction (PCR)
Thermocyclers
Components:
-Water
Template DNA
-Nucleotides (A, T, G, C)
-Magnesium Chloride/ Buffer
-A set of primers per locus
-Taq Polymerase
Assemble in a reaction tube/plate
Place in Thermocycler
95°C …30 sec62°C …30 sec72°C …1min
25-40 cycles
http://www.youtube.com/watch?v=2KoLnIwoZKU
Source: Wikipedia
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1,048,555
After just 20 PCR cyclesYou will have:
carbon copies of the DNA from that specific area in the genome
Polymerase Chain Reaction (PCR)Source: J. Butler
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Need between 0.1 to 2.5 ng of DNA to get a full profile with
a PCR-based DNA test
1 GRAM (~ 1/25th of an ounce)
= 1,000 mg (miligrams)
= 1,000,000 μg (micrograms)
= 1,000,000,000 ng (nanograms)
= 1,000,000,000,000 pg (picograms)
Polymerase Chain Reaction (PCR)
Each cell has about 6.5 picograms of DNA on average
Highly sensitive
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Chr 8(D8S1179)
Chr 21(D21S11)
Chr 7(D7S820)
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Polymerase Chain Reaction (PCR)
Capillary Electrophoresis (CE)
Cap
illar
y
PCR Tube or 96-well plate
Data Acquisition
Det
ecto
r
Modified with permissionfrom: Butler, J; NIST
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Old style Gels
New World electropherograms
Source: PD
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CE Instrument: 310, 3100, 3130
Capillary Electrophoresis (CE)
Source: PD
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Markers
There are more than 100,000 STR markers in the human genome
STR Loci in Modern ForensicsSource: NIST
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Electropherograms or “e-grams”
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1
2
3
4
Modern DNA Profiling
Locus allele1 allele2 D18S1179 13
D21S11 30
D7S820 10 11CSF1PO 10 12D3S1358 14 15TH01 8 9.3D13S317 11D16S539 11 12D2S1338 19 23D19S433 14 15 VWA 17 18TPOX 8D18S51 15 19Amelogenic XD5S818 11FGA 23 24
DNA Profile of
“Jane Doe”
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Identifiler DNA Testing Kit from
ABI/Life Technologies
Markers not always included in statistical calculations
Electropherogram
RFUOr Peak Height (1336) Allele Calls for
Marker TH01 (8 , 9.3)
RFU=Relative Fluorescence Units
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Balance
Stutter
Artifacts / Anomalies / Challenges
Biologically InducedTechnically Induced
Split Peaks
Microvariants
Dye Blobs
Pull-Ups
Spikes
Tri-alleles
Allele Drop In / OutAllele Peak Imbalance
Sample Induced
Contamination
Mixtures
Inhibition
Low Copy Number DNA (LCN)
Increased Complexity
Wrong Conclusions
Complex MixturesStutters 49
Allele 8
Dye Blobs
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Dye Molecule
Pull‐Up Peaks
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Data on panel 1
Data on panel 2
Pull-up
Spike Peaks
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Spikes
Panel 1
Panel 2
Panel 3Panel 4
Allele 7Stutter
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Most Copies are of Allele 7
- 1 Stutter (“allele 6”)
Stut
ter
+ 1 Stutter (“allele 8”)
Allele 11
Stutter
1110 12
<15% <5%
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Evidence Sample
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Evidence Sample
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Evidence Sample
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Eviden
ce Sam
ple
“Dropouts”
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?
Evidence Sample
Is allele 12 a stutter peak?
DNA Analysis: Steps
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-Sample Collection
-DNA Extraction
-Quantitation
-Amplification
-Electrophoresis
-Data Interpretation
Sample Types
Positive Controls
Negative Controls
Evidence Samples (Qs)
Reference Samples (Ks)
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DNA Analysis: Steps
Sample Collection
DNA Extraction
DNA Quantitation
Amplify w/ PCR
Capillary Electrophoresis
Results
Results Interpretations / Statistics
Optimum amount is 1 nanogram of DNA
Re-PCR
Increase Injection time
Contamination?
Re-inject63
• Match – Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significance of the match is usually reported with the match report.
• Exclusion (Non‐match) – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.
• Inconclusive – The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.
Outcomes of Forensic DNA Analysis
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Allele Frequency in the Population
Frank Judy
Anna Paul Tony Phil
8 4 8 7
7813612131414
Liz
8 12
Michelle
8 12
Allele Frequency in the Population
Allele Name
Size of “Population” Surveyed
# Chrom. Detected in Population
Allele Frequency
Approx%
Allele 4 16 1 0.0625 6%
Allele 6 16 1 0.0625 6%
Allele 7 16 2 0.125 12%
Allele 8 16 5 0.3125 31%
Allele 12 16 3 0.1875 18%
Allele 13 16 2 0.125 12%
Allele 14 16 2 0.125 12%
Total = 1 or 100%
Locus allele value allele value 1 in CombinedD3S1358 16 0.2533 17 0.2152 9.17 9.17
VWA 17 0.2815 18 0.2003 8.87 81
FGA 21 0.1854 22 0.2185 12.35 1005
D8S1179 12 0.1854 14 0.1656 16.29 16,364
D21S11 28 0.1589 30 0.2782 11.31 185,073D18S51 14 0.1374 16 0.1391 26.18 4,845,217D5S818 12 0.3841 13 0.1407 9.25 44,818,259D13S317 11 0.3394 14 0.0480 30.69 1.38 x 109
D7S820 9 0.1772 31.85 4.38 x 1010
D16S539 9 0.1126 11 0.3212 13.8 6.05 x 1011
THO1 6 0.2318 18.62 1.13 x 1013
TPOX 8 0.5348 3.50 3.94 x 1013
CSF1PO 10 0.2169 21.28 8.37 x 1014
The Random Match Probability for this profile in the U.S. Caucasian population is 1 in 837 trillion (1012)
Where do these Astronomical Numbers come from?
What is it? Forensic Serology
Blood
Saliva
Semen
Urine….& others
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Presumptive Tests: Confirmatory Tests:
DNA
“Touch” DNA
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What is “touch” DNA?
-DNA from potential contact with human skin.-Skin cells, Sweat, and oil from skin glands (cell-free DNA).
-Term is used to describe the nature of sample being analyzed.
-Typically:-Swabs from surfaces or items like weapons, stolen goods, etc.-Samples usually contain small amounts of DNA.-Analysis can yield partial DNA profiles.
-The word “touch” can be misleading.
-Analysis cannot prove whether contact was direct or indirect.
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Forensic DNA evidence cannot tell you how, when, and under what circumstances a DNA sample was left at the crime scene.
Keep in Mind:
DNA evidence is very powerful. It is the best forensic tool available so far to identity a potential perpetrator.
DNA evidence cannot reveal the source of DNA (blood, semen, etc). DNA from blood will look exactly like DNA from skin.
DNA evidence can be weak and misleading, and the interpretation of that evidence can be highly subjective.
-The Forensic Laboratory and Quality Assurance Standards (QAS)
Future Webinar Topics:
-Statistics: Understanding the Weight of DNA Evidence
-Advanced Topics in Forensic DNA Testing:
Mixture Interpretations
Mitochondrial DNA Analysis
Y-STR DNA Analysis
-Forensic Serology: Bodily Fluids Identification
“Touch” DNA
Where is this “touch” DNA coming from??
KeratinizationBody Secretions
Things to Keep in Mind:
Cells
Secondary Transfer
PhysiologySurface
SWAB
Free DNA72
The Forensic Laboratory and Quality
Assurance Standards (QAS)
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TWGDAM (1988)
SWGDAM (2000)QAS (1998)
FBI Laboratory:
Your State or LE Forensic Lab
Internal AuditsExternal Audits(CW and DB)
Accreditation(ASCLD-LAB, FQS, ISO)
2007 -2004-2007-2010QAS (2009)
ISO
ISFG
NIST
AAFS
DNA Testing Standards and Quality Assurance
Literature
Literature
Lite
ratu
re
CongressionalDNA Act (1994).
NFSTC
DNA Advisory Board
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-Laboratory Accreditation / Audits
-Laboratory Management / Case Management
-Staff Proficiency / Education
-Validation of instruments, test kits, protocols
-Quality Control, sample prep, processing, integrity
-Results and Interpretations Standards
Quality Assurance Standards
The Laboratory “Should” establish most standards
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Quality Assurance Standards
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Source: www.nist.gov
Lab Discovery
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Lab reports: Final as well as any/all rescinded / cancelled reports
Evidence Submission Forms
Full Case File (will vary slightly based on the laboratory)
Serology Analysis / Analyst Notes
DNA Work Sheets / Analyst Notes : -DNA Extraction-Quantitation-Amplification -Electrophoresis (e-grams and raw data if needed)-Allele calls/Data tables-Statistical analyses-Controls
Evidence Packaging / Analyst Notes
Lab Discovery—cont.
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Case-related e-mails and communication
Photographs of evidence (if any)
Chain of Custody documents
Contemporaneous and recent revision ofSOPs and protocols for analysis and interpretation
Analyst CV
Quality Assurance Records:-QA Manual-Lab accreditation and audits-Non conformity records-Corrective Action records
Technical and Administrative reviews
CODIS submission documents
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Preservation of Evidence
Questions
Thank you:
For Questions or Comments:
Please send an e-mail to Dr. Max Noureddine: